Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while differences in structure between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my studies focus on: 1) identifying the earliest events which occur during prion infection, 2) precisely defining the different cellular compartments where PrPSc formation occurs, 3) determining the molecular basis of prion strains and, 4) development of effective prion therapeutics. One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrPSc, but it is unclear how the different biochemical properties of PrPSc influence its uptake. Historically, it has always been believed that the infectious prion particle consisted mainly of highly protease-resistant PrPSc. Recently, however, it has become apparent that not all infectious PrPSc is highly protease-resistant. There are forms of PrPSc which are relatively protease-sensitive that are present in stoichiometric amounts with protease-resistant PrPSc. This ratio of protease-sensitive to protease-resistant PrPSc can vary depending upon the prion isolate, with some infectious isolates consisting of mostly protease-sensitive PrPSc. Protease-sensitive PrPSc molecules are thought to contribute to different aspects of prion pathogenesis in vivo such as disease incubation times, although the mechanisms are unclear. In particular, there are no studies looking at how protease-sensitive PrPSc interacts with cells when compared to protease-resistant PrPSc. In 2014, we completed and published experiments characterizing the interaction of protease-sensitive versus protease-resistant forms of PrPSc with human astrocyte cells during the initial stages of human prion infection. We showed that, for 4 different types of human prions, protease-sensitive PrPSc was internalized and degraded by the cell similarly to protease-resistant PrPSc. This work is in press at The American Journal of Pathology. In 2014, we continued in vivo work to look at early events during prion infection following intracranial inoculation. These studies are designed to examine the early formation of PrPSc as well as changes in the expression of the host PrPC molecule during the acute stage of prion infection. Our work will provide critical information about the events that occur during the first few hours following exposure to prions, including when formation of new PrPSc occurs and how PrPC is affected. In 2014, we have begun using a newly developed in vitro system to study the effect of PrPC amino acid polymorphisms on human PrPSc formation. We are also utilizing both cell-free and cell-based systems to assess human PrPSc formation and how different human prion strains interact with different cell types to trigger infection. In 2014 we continued our collaboration with Dr. Mark Head and Dr. James Ironside at the CJD Surveillance unit in Edinburgh, Scotland. This collaboration involves using proteomics to study different human prion strains. These studies will provide insight into the pathogenesis and classification of different human prion diseases. Finally, in 2014 we have initiated preliminary studies to test whether or not inhibitors of neuroinflammation can also inhibit or delay prion disease.